We have focused attention on the 1.2 kb insulator DNA sequence at the 5 end of the chicken beta-globin locus, and elements upstream of it. This insulator is capable both of blocking the influence of outside enhancers and of preventing the encroachment of condensed chromatin that might shut down expression of the entire region. We have shown previously that enhancer blocking activity is associated with binding of a single protein, CTCF, to a site within the enhancer. We have shown that this protein is responsible for regulation of imprinted gene expression at several imprinted loci. In order to understand the mechanism of action of CTCF we have determined the minimum CTCF fragment that can bind to its target DNA site with full affinity. We have also continue to extend our earlier studies showing that CTCF molecules interact with one another. This provides important information for design of proteins that can modulate CTCF function within the nucleus. Among the interactions now under investigation is the interaction of CTCF with the cohesin protein complex, recently shown in the laboratories of Matthias Merkenschlager and others to be essential to the recruitment of cohesin to DNA at some stages of the cell cycle. We are collaborating with Dr. Merkenschlager in dissecting this interaction. Our recent results have identified both the cohesin subunit that directly interacts with CTCF, and the domain of CTCF with which it interacts. We have also used mass spec analysis to identify other co-factors that appear to be important for CTCF insulator function. Using these techniques, we have also identified a novel and important co-factor of CTCF, the regulatory protein p68. We have shown that this protein, together with its associated RNA co-factor,binds to CTCF, is present genome-wide at CTCF occupies sites on chromatin, and is essential for insulator activity.